Pharmaceutical formulations of xanthogenates and inhibitors of viral nucelic acid replication

ABSTRACT

The invention relates to pharmaceutical formulations of xanthogenates and agents containing these formulations for the treatment of viral, tumor or autoimmune diseases. The pharmaceutical formulations contain a xanthogenate of formula I  
                 
 
wherein R 1  represents an optionally substituted aryl or alkyl residue and R 2  represents a metal atom, an optionally substituted alkyl, alkoxy, amino or ammonium group or halogen, and an inhibitor of viral nucleic acid replication such as e.g. aciclovir, valaciclovir, penciclovir, famciclovir, and optionally a carrier substance reducing the irritating effect of the xanthogenate and optionally an adjuvant enhancing the activity.

The invention relates to pharmaceutical formulations of xanthogenates incombination with inhibitors of viral nucleic acid replication and agentscontaining these formulations for the treatment of viral diseases.

Xanthogenates, in particular tricyclodecane-9yl-xanthogenate (D609), areknown as substances with antiviral and antitumor activity, e.g. from“DNA and RNA virus species are inhibited by xanthates, a class ofantiviral compounds with unique properties” Sauer-G; Amtmann-E;Melber-K; Knapp-A; Muller-K; Hummel-K; Scherm-A, inProc-Natl-Acad-Sci-U-S-A. 1984 June; 81(11): 3263-7; “Selective killingof tumor cells by xanthates” Amtmann-E; Sauer-G, in Cancer-Lett. 1987June; 35(3): 237-44, and U.S. Pat. No. 4,602,037.

The pharmaceutical utilization of xanthogenates with antiviral andantitumor activities is impeded by the fact that relatively high agentconcentrations are required in order to demonstrate efficacy in ananimal model. However, since the concentration of the agent is limitedboth for pharmacological and technical reasons, even the maximal usableconcentrations achieve only a limited healing effect. The same problemapplies to common antiviral inhibitors, such as aciclovir, valacicloviror famciclovir.

We have surprisingly found that a synergistic enhancement of the effectis obtained by combining xanthogenate derivatives, such as D609, andinhibitors of viral nucleic acid replication, such as aciclovir. In thepresence of low, antivirally ineffective concentrations of thexanthogenate, the effect of aciclovir in cell culture was observed to beincreased up to five-fold. In animal experiments, the combination ofD609 and aciclovir achieved survival of all HSV-1-infected animals. Eachactive ingredient applied separately achieved only partial healing.

The present invention thus solves the aforementioned problem byproviding a pharmaceutical formulation that contains a xanthogenate andan inhibitor of viral DNA or RNA replication.

The formulation contains a xanthogenate of general formula I

wherein R₁ represents an optionally substituted aryl or alkyl residue.

R₁ preferably represents an adamantyl, norbornyl, tricyclodecyl, benzyl,linear or branched C₃-C₂₀ alkyl, C₃-C₂₀ cycloalkyl, furyl, pyridyl,anthracyl, naphthyl, phenanthryl, perinaphthyl or quinuclidinyl residue,whereby the aforementioned linear or branched C₃-C₂₀ alkyl residue canbe substituted by a hydroxyl, a C₁-C₄ alkoxy group, a halogen atom or anamino group, and the aforementioned C₃-C₂₀ cycloalkyl residue can alsobe substituted by a hydroxyl, a C₁-C₄ alkoxy or a C₁-C₄ alkyl group, ahalogen atom or an amino group. Particularly preferred for R₁ arecyclododecyl, dodecyl, undecyl, decyl, tricyclo[5,2,1,0^(2,6)]-decyl,nonyl, octyl, bicyclo[2,2,1]-heptyl, cyclohexyl, hexyl, toluyl residues.Particularly advantageous is an exo/exo-tricyclo[5.2.1.0^(2,6)]-decylresidue.

R₂ represents a metal atom, an optionally substituted alkyl, alkoxy,amino or ammonium group or halogen. Preferably, R₂ represents a mono- ormultivalent metal atom, a linear C₁-C₆ alkyl residue, ahydroxy-substituted C₁-C₆ alkyl residue, a C₁-C₆ alkoxy residue, anamino group, a C₁-C₆ alkylamino residue, a di-(C₁-C₆ alkyl)aminoresidue, a tri-(C₁-C₆ alkyl)ammonium residue, a halogen,2,3-dihydroxypropyl or hydroxy-(C₁-C₆ alkoxy)methyl. Particularlyadvantageous are sodium and potassium salts and dimethylglycyl andmethyl esters.

Preferably, the inhibitor of viral nucleic acid replication is anucleoside analogue, and particularly advantageously it isbromodeoxyuridine (BudR), fluorodeoxyuridine (FudR), aciclovir,valaciclovir, penciclovir or famciclovir.

The inhibitor of viral nucleic acid replication can also be an inhibitorof viral helicase.

The inhibitor of viral nucleic acid replication can also be an inhibitorof a cellular enzyme.

Formulations containing 0.1 to 10 parts of inhibitor of viral nucleicacid replication per one part of xanthogenate have proven to beespecially useful. Particularly advantageous is axanthogenate-to-inhibitor of viral nucleic acid replication ratio of1:1.

The formulation according to the invention preferably contains, inaddition, an ionic detergent as an effect-enhancing adjuvant such as isdescribed in U.S. Pat. No. 4,851,435. A fatty acid with 6-19 C atoms orits salt is particularly preferred as adjuvant. Particularly preferredare potassium salts of decanoic, undecanoic or lauric acid. Theactivity-enhancing adjuvant can also be a sulphate with an aliphaticresidue of 8-18 C atoms. Particularly preferred is sodium lauric acidsulphate. Moreover, deoxycholic acid or a pharmaceutically tolerablesalt thereof or a phosphonic acid can be used as the adjuvant.

Moreover, it is preferred to incorporate the xanthogenate in lipid- orsteroid-based carrier substances in accordance with WO 96/14841.Incorporation into a carrier substance improves the tolerability of theagents. In this context, the carrier substance is particularly a steroidsuch as cholesterol, cholestanol, cholanic acid, chondrillasterol, andα, β, γ sisterol.

It is particularly preferred for xanthogenate and adjuvant, if any, tobe mixed with the carrier substance in accordance with DE 101 17 728.Cholesterol is particularly preferred. Phospholipids, in particularphosphatidylcholine, phosphatidylserine, phosphatidylinositol orstearylamine are also suited for use as carrier substance.

A formulation containing aciclovir, the Na or K salt of decanoic acid,and exo/exo-tricyclo[5,2,1,0^(2,6)]-9yl-xanthogenate is particularlypreferred. In particular, this formulation contains one partxanthogenate, one part potassium salt of decanoic acid, and one partaciclovir.

Another particularly preferred formulation contains phosphatidylcholineor cholesterol, the Na or K salt of decanoic acid,exo/exo-tricyclo[5,2,1,0^(2,6)]-9yl-xanthogenate, and aciclovir. Inparticular, this formulation contains one part xanthogenate, one partdecanoic acid, four parts phosphatidylcholine or cholesterol, and onepart aciclovir.

According to claims 11 to 16, the present invention also providesagents, containing the pharmaceutical formulation for the treatment ofviral, tumor or autoimmune diseases. In addition, these agents containcommon carrier substances and/or common excipients. It is also possiblethat other active ingredients are contained therein provided theyinterfere neither with the effect nor the stability of the xanthogenatesand inhibitors of viral nucleic acid replication.

Particularly preferred are agents in the form of ointments, whereby alipophilic substance is used as the ointment base. Preferably, vaselineis used as the ointment base.

The pharmaceutical formulation according to the invention and agentscontaining them are suitable for the treatment of viral, tumor, andautoimmune diseases.

The following examples illustrate the invention in more detail withoutlimiting the invention.

EXAMPLE 1

Synergistic Enhancement of the Inhibitory Effect of Aciclovir on theProliferation of Herpes Simplex Virus by the exo/exo Isomer oftricyclo[5,2,1,0^(2,6)]-9yl-xanthogenate.

Human lung carcinoma cells (Calu-6) were infected with 30 plaque-formingunits of herpes simplex virus (Type-1, strain ANG) in Linbro plates.Cell culture medium (MEM containing 10% fetal calf serum, 0.85 g/lsodium bicarbonate, and 0.5% carboxymethylcellulose) containing either0, 5 or 10 μg/ml exo/exo-tricyclo[5,2,1,0^(2,6)]-9yl-xanthogenate (D609)was added two hours after infection. The cultures were treated withaciclovir at the same time. All samples were processed asquadruplicates. The cells were incubated for 48 h at 37° C. under CO₂gassing (5%). Then the medium was decanted, the cells were fixed with 3%formalin and stained with 0.5% crystal violet. After drying at roomtemperature, the number of plaques formed was determined.

In the cultures containing no D609 the number of plaques counted was32.75±11. In the presence of 5 or 10 μg/ml D609 the number of plaquescounted was 32±3 plaques and 33±6 plaques, respectively. This means thatD609 had no effect on plaque formation at these concentrations. In thepresence of aciclovir at a concentration of 0.16 μM the number ofplaques was reduced to 30±11. This effect was not statisticallysignificant (student's t-test p=0.4).

In the presence of 10 μg/ml D609 and 0.16 μM aciclovir, the number ofplaques was reduced to 9.5±5.1. This effect was statisticallysignificant (student's t-test p=0.014).

The results are illustrated in FIG. 1, in which the mean number ofplaques is plotted over the aciclovir concentration. The series ofmeasurements without D609 is indicated by squares, the series ofmeasurements with 5 g/ml D609 by cycles, and the series of measurementswith 10 g/ml D609 by triangles. It is clearly evident that the presenceof D609 at concentrations that are ineffective by themselves providesfor the onset of the effect of aciclovir at substantially lowerconcentrations, at which aciclovir alone does not show an effect.

EXAMPLE 2

Enhancement of the Effect of Aciclovir on the Course of ExperimentalInfection with Herpes Simplex Virus in Mice by the exo/exo Isomer oftricyclo[5,2,1,0^(2,6)]-9yl-xanthogenate.

One part (by weight) D609, one part (by weight) potassium salt ofdecanoic acid, and four parts (by weight) cholesterol were mixed in amortar. Subsequently, propyleneglycol (final concentration 10%) andvaseline were added such that the final concentration of D609 was 5%.The same procedure was used to produce ointments containing 5% acicloviror 5% D609 and 5% aciclovir. A placebo ointment containing neither ofthe two agents was produced using the analogous procedure.

Ten mice (Balb-C strain) each were shaved on their upper leg and sixscratches in the skin were made with a cannula in an area of 5×5 mm.Subsequently, a cotton bud was used to apply 50 μl of a HSV-1 suspension(Wal strain, 10⁸ plaque-forming units/ml). The treatment (twice daily)was initiated four days after infection. The symptoms, “widespreadlesions”, “hind leg paralysis”, and survival were recorded in theprotocol.

The result is shown graphically in FIGS. 2 a to 2 d. The plot shows thenumber of animals displaying the symptoms indicated over the number ofdays after infection. Triangles indicate the surviving animals in eachcase, squares the animals with hind leg paralysis, and diamonds theanimals with widespread lesions.

FIG. 2 a shows for treatment with the placebo ointment containing noagent that only 3 animals survived after 14 days and that all animalshad shown widespread lesions and 7 animals had shown paralysis. In FIGS.2 b and 2 c, treatment with D609 (2 b) or aciclovir (2 c) is shown torender the survival rate higher, and lesions and paralysis almostcompletely healed. For the combination of D609 and aciclovir, FIG. 2 dshows that all animals survived and lesions and paralysis werecompletely healed and manifested to a lesser degree.

Accordingly, the combination preparation is clearly more efficaciousthan the individual preparations. The combination preparation achievessurvival of all animals and a lesser degree of symptom manifestation andclearly more rapid healing of symptoms.

1. Pharmaceutical formulation, comprising a xanthogenate of formula I

 wherein R₁ represents an optionally substituted aryl or alkyl residue,and R₂ represents a metal atom, an optionally substituted alkyl, alkoxy,amino or ammonium group or halogen, an inhibitor of viral nucleic acidreplication, optionally an adjuvant enhancing the activity of thexanthogenate, and optionally a carrier substance reducing the irritatingeffect.
 2. Pharmaceutical formulation according to claim 1, wherein R₁is an adamantyl, norbornyl, tricyclodecyl, benzyl, linear or branchedC₃-C₂₀ alkyl, C₃-C₂₀ cycloalkyl, furyl, pyridyl, anthracyl, naphthyl,phenanthryl, perinaphthyl or quinuclidinyl residue, whereby the saidlinear or branched C₃-C₂₀ alkyl residue can be substituted by ahydroxyl, a C₁-C₄ alkoxy group, a halogen atom or an amino group, andthe said C₃-C₂₀ cycloalkyl residue can be substituted by a hydroxyl, aC₁-C₄ alkoxy, a C₁-C₄ alkyl group, a halogen atom or an amino group. 3.Pharmaceutical formulation according to claim 2, wherein R₁ is acyclododecyl, dodecyl, undecyl, decyl, tricyclo[5,2,1,0^(2,6)]-decyl,nonyl, octyl, bicyclo[2,2,1]-heptyl, cyclohexyl, hexyl or toluylresidue.
 4. Pharmaceutical formulation according to claim 1, wherein R₂is a sodium or potassium atom or a dimethylglycylester or methylestergroup.
 5. Pharmaceutical formulation according to claim 1, wherein theinhibitor of viral nucleic acid replication is a nucleoside analogue. 6.Pharmaceutical formulation according to claim 5, wherein the inhibitorof viral nucleic acid replication is selected from aciclovir,valaciclovir, pencidovir, and famciclovir.
 7. Pharmaceutical formulationaccording to claim 1, comprising 1 to 10 parts inhibitor of viralnucleic acid replication per one part xanthogenate.
 8. Pharmaceuticalformulation according to claim 1, comprising an ionic detergent asadjuvant.
 9. Pharmaceutical formulation according to claim 1, comprisingdeoxycholic acid or a pharmaceutically tolerable salt thereof asadjuvant.
 10. Pharmaceutical formulation according to claim 1,comprising a phosphonic acid as adjuvant.
 11. Pharmaceutical formulationaccording to claim 1, comprising cholesterol as carrier substance. 12.(canceled)
 13. Pharmaceutical formulation according to claim 1, whereinthe xanthogenate is tricyclo[5,2,1,0^(2,6)]-decane-9-yl-xanthogenate,the carrier substance is cholesterol or phosphatidylcholine, theadjuvant is the sodium or potassium salt of decanoic acid, and theinhibitor of viral nucleic acid replication is selected from aciclovir,valaciclovir, penciclovir, and famciclovir.
 14. Pharmaceuticalformulation according to claim 13, wherein the inhibitor of viralnucleic acid replication is aciclovir.
 15. Pharmaceutical formulationaccording to claim 1, comprising one part xanthogenate, one partinhibitor of viral nucleic acid replication, four parts carriersubstance, and one part adjuvant.
 16. Pharmaceutical formulationaccording to claim 1, further comprising a lipophilic substance asexcipient for administration as an ointment.
 17. Pharmaceuticalformulation according to claim 16, wherein the lipophilic substance isvasoline.
 18. Pharmaceutical formulation according to claim 7,comprising 2 to 4 parts inhibitor of viral nucleic add replication perone part xanthogenate.
 19. Pharmaceutical formulation according to claim8, wherein the ionic detergent is a fatty add with 6 to 19 C atoms or analkylsulphate with 8 to 18 C atoms.
 20. Method for the treatment ofviral, tumor, or autoimmune diseases comprising administering to apatient in need thereof an effective amount of a xanthogenate of formulaI

wherein R₁ represents an optionally substituted aryl or alkyl residue,and R₂ represents a metal atom, an optionally substituted alkyl, alkoxy,amino or ammonium group or halogen, and an effective amount of aninhibitor of viral nucleic acid replication.
 21. Method according toclaim 20, wherein R₁ is a cyclododecyl, dodecyl, undecyl, decyl,tricyclo[5,2,1,0^(2,6)]-decyl, nonyl, octyl, bicyclo[2,2,1]-heptyl,cyclohexyl, hexyl or toluyl residue and R₂ is a sodium or potassium atomor a dimethyl-glycylester or methylester group.
 22. Method according toclaim 20, wherein the inhibitor of viral nucleic acid replication isselected from aciclovir, valaciclovir, penciclovir, and famciclovir. 23.Method according to claim 20, further comprising cholesterol orphosphatidylcholine as a carrier substance, and sodium or potassium saltof decanoic acid as an adjuvant, and wherein the xanthogenate istricyclo[5,2,1,0^(2,6)]-decane-9-yl-xanthogenate, and the inhibitor ofviral nucleic acid replication is selected from aciclovir, valaciclovir,penciclovir, and famciclovir.